Volume 67, Issue 2, Pages 349-359 (August 2017) Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis  Ji Yeon Kim, Keon Jae Park, Joo-Yeon Hwang, Gyu Hee Kim, DaeYeon Lee, Yoo Jeong Lee, Eun Hyun Song, Min-Gyu Yoo, Bong-Jo Kim, Young Ho Suh, Gu Seob Roh, Bin Gao, Won Kim, Won-Ho Kim  Journal of Hepatology  Volume 67, Issue 2, Pages 349-359 (August 2017) DOI: 10.1016/j.jhep.2017.03.023 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 1 ATF3 overexpression in the livers of ZDF rats and human participants with NAFLD. (A) Imaging and quantification of ATF3 expression in the livers of ZDF rats (n=8). (B) Immunostaining analyses of ATF3 (ICC) and 4-HNE (IHC) in liver tissues of 19-week-old rats (100×; n=8, each group). (C) An integrative functional significance of ATF3 in NAFLD-related gene network. Red and blue colors indicate the genes that were closely linked to ATF3, which are upregulated and downregulated, respectively, in human livers with NAFLD. (D) Hepatic expression of ATF3 (left) and FAS mRNA levels (middle) in participants with no NAFLD (NAS=0), mild NAFLD (NAS 1–5), and morbidly obese NAFLD (NAS >5) from a public database (GSE48452) and their correlation analyses (right). (E and F) Correlation between ATF3 and JUN mRNA levels (E; p=0.421 between NAS=0 vs. NAS 1–5) or serum adiponectin levels (F; p=0.513 between NAS=0 vs. NAS 1–5). (A and B) All data are given as the mean±SEM with *p<0.01 and **p<0.05 from the t test. (D, E, and F) Data are presented as box plots (median, IQR). The top and bottom of each box indicate the 1st and 3rd quartiles (interquartile range, IQR), and the band inside the box indicates the median. The dot indicates an outlier more than 1.5 times the IQR below the 25th percentile or more than 1.5 times the IQR above the 75th percentile of all data. p values in D–F from post hoc analysis for the ANOVA; p<0.001 form the ANOVA. ZL, Zucker lean; ZDF, Zucker diabetic fatty; ATF3, activating transcription factor 3; 4-HNE, 4-hydroxy-2-nonenal; ICC, immunocytochemistry; IHC, immunohistochemistry; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; FAS, fatty acid synthase; SEM, standard error of the mean; ANOVA, analysis of variance. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 2 In vivo ATF3 silencing ameliorates impaired glucose metabolism and hepatic steatosis in ZDF rats. Either in vivo-jetPEI ATF3 siRNA (A3 si) or scrambled siRNA (Scr.) was intravenously administered to 15-week-old ZL or ZDF rats (n=8, each group). (A) Oral glucose tolerance test (OGTT). (B) Correlation between ATF3 protein expression and liver TG levels or liver/body weight ratios. (C) Representative Western blots and densitometric quantification relative to IRS-1. (D) Hepatic expression of lipogenesis-related proteins. (E) Hematoxylin and eosin (H&E) staining and IHC analysis (100×) of SREBP-1c and FAS proteins and staining hepatocytes were scored. All data are given as the mean±SEM with *p<0.01 and **p<0.05 from the t test. ZL, Zucker lean; ZDF, Zucker diabetic fatty; ATF3; activating transcription factor 3; TG, triglyceride; IRS-1, insulin receptor substrate 1; pY (941), active IRS-1 (Tyr-941) phosphorylation; pS (307), inhibitory IRS-1 (Ser-307) phosphorylation; IHC, immunohistochemistry; SREBP-1c, sterol regulatory element binding protein 1c; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 3 ATF3 silencing counteracts impaired fatty acid oxidation and metabolic rates in ZDF rats. (A) Hepatic expression of proteins related to fatty acid oxidation and lipid accumulation. (B) CPT-1 activity in the mitochondria of primary hepatocytes isolated from rats. (C) Total liver and mitochondrial β-oxidation rates. (D) Correlation between the ATF3 protein expression and β-oxidation rates. (E) Real-time PCR analyses for hepatic mRNA expression of fatty acid uptake (left), synthesis/lipogenesis (middle), and oxidation (right) markers. (F) The lactate/pyruvate ratio in the livers of ZDF rats and the NADH/NAD+ ratio were determined. All data are given as the mean±SEM with *p<0.01 and **p<0.05 from the t test (n=8, each group). ZL, Zucker lean; ZDF, Zucker diabetic fatty; PGC-1α, PPAR-γ coactivator 1α; PPAR-α, peroxisome proliferator-activated receptor α; CPT-1, carnitine palmitoyltransferase 1; PPAR-γ, peroxisome proliferator-activated receptor γ; FSP27, fat-specific protein 27; CD36, fatty acid translocases; FATP2, fatty acyl transport protein 2; FABP1, fatty acid binding protein 1; ACSL1, acyl-CoA synthetase long-chain family member 1; ACC, acetyl CoA carboxylase; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1; DGAT1, diglyceride acyltransferase 1; SREBP1, sterol regulatory element binding protein 1; SPT1, serine palmitoyltransferase 1; ACOX1, acyl-coenzyme A oxidase 1; LCAD, long-chain fatty acyl-CoA dehydrogenase; SEM, standard error of the mean. Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 4 In vivo ATF3 silencing inhibits the hepatic inflammatory responses in ZDF rats. Either 100 or 150μg of ATF3 siRNA was administered to 15-week-old ZL or ZDF rats (n=8, each group). (A) Serum pro-inflammatory cytokines. (B and C) Real-time PCR analyses of hepatic mRNA expression of pro-inflammatory cytokines (B) and chemokines (C). (D) IHC analysis for CD68 (100x) and the quantification of CD68-positive cells. (E) Chemotaxis analysis for macrophage migration. (F) ATF3 silencing reduces NF-κB activation in ZDF rats. All data are given as the mean±SEM with *p<0.01 and **p<0.05 from the t test. ATF3, activating transcription factor 3; Scr. si, scrambled siRNA; ATF3 si, ATF3 siRNA; ZL, Zucker lean; ZDF, Zucker diabetic fatty; IL-6, interleukin 6; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein 1; IL-1, interleukin 1; MIP-1, macrophage inflammatory protein 1; ICAM1, intercellular adhesion molecule 1; Col4, collagen type IV; SOCS, suppressor of cytokine signaling; CCR2, chemokine (C-C motif) receptor 2; CXCL2, chemokine (C-X-C motif) ligand 2; TNFR, tumor necrosis factor receptor; CTL, control; RANTES, regulated on activation, normal T cell expressed and secreted; IHC, immunohistochemistry; SEM, standard error of the mean. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 5 NAFLD is closely associated with T2D. (A) Comparison of diabetes-related, biochemical parameters in participants with and without NAFLD (n=322). (B) The histologic grades of steatosis by H&E staining in liver biopsy tissues. (C) Comparison of the prevalence (%) of diabetes in participants with or without NAFLD (p value was determined by the chi-squared test). (D) Comparison of diabetes-related, biochemical parameters between participants with NAFLD alone and those with NAFLD plus diabetes. (A and D) Data are presented as box plots (median, IQR) and p value was determined by the t test. FBG, fasting blood glucose; HOMA-IR, the homeostasis model assessment of insulin resistance; NAFLD, non-alcoholic fatty liver disease; DM, diabetes mellitus. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 6 ATF3 induction correlates with hepatic steatosis in NAFLD participants. Fifty liver biopsy samples with or without steatosis were randomly selected. (A) Real-time PCR for ATF3 mRNA in the livers of controls (n=7) and participants with steatosis grades 1 (n=13), 2 (n=17), and 3 (n=13). (B) Real-time PCR for the hepatic mRNA levels in the livers of NAFLD participants. All data are given as the mean±SEM with *p<0.05, **p<0.005, and ***p<0.0005 from the t test. (C) Correlation analysis based on real-time PCR data. Correlation coefficients (r) and p values are given. (D) IHC analysis and quantification of ATF3 in liver tissues across steatosis grades. Data are presented as box plots (median, IQR) (*p<0.05 and **p<0.005 vs. steatosis grade 0 from the t test). (E) IHC analysis of CHOP and SREBP-1c in liver tissues with low (n=18) and high (n=32) expression levels of ATF3. The expression scores were assessed based on their intensity and distribution, and they were divided into three groups: low (0–1.49), middle (1.50–2.99), and high (≥3). ATF3, activating transcription factor 3; NAFLD, non-alcoholic fatty liver disease; SREBP1c, sterol regulatory element binding protein 1c; FAS, fatty acid synthase; PPAR-γ, peroxisome proliferator-activated receptor γ; PPAR-α, peroxisome proliferator-activated receptor α; PGC-1α, PPAR-γ coactivator 1α; CHOP, CCAAT-enhancer-binding protein homologous protein; MCP-1, monocyte chemoattractant protein 1; IHC, immunohistochemistry; SEM, standard error of the mean. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 7 ATF3 induction precedes macrophage infiltration and diabetes in NAFLD participants. (A) IHC analysis and quantification of CD68-positive cells in human liver tissues (*p<0.01 vs. steatosis grade 0 from the t test). (B) Correlation of an ATF3 staining score with CD68-positive cells in NAFLD participants. (C) Comparison of ATF3 mRNA and protein expression among controls, participants with NAFLD alone, and those with NAFLD plus diabetes (p<0.001 and p<0.005, respectively from post hoc analysis for the ANOVA; p<0.001 from the ANOVA). (D) Comparison of hepatic ATF3 mRNA expression in NAFLD participants with (HOMA-IR ≥2) and without (HOMA-IR <2) insulin resistance (p<0.05 from the t test). (E) The prevalence (%) of diabetes with or without insulin resistance among the participants with NAFLD (p value was determined by the chi-squared test). (A, C, and D) Data are presented as box plots (median, IQR). ATF3, activating transcription factor 3; NAFLD, non-alcoholic fatty liver disease; DM, diabetes mellitus; HOMA-IR, the homeostasis model assessment of insulin resistance. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 349-359DOI: (10.1016/j.jhep.2017.03.023) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2017 67, 349-359DOI: (10. 1016/j. jhep. 2017. 03 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions